This proposal will test the hypothesis that upregulation of the HEF1 gene in tumors is an important contributing factor for cancer metastasis, based on the ability of elevated HEF1 to drive epithelial-mesenchymal transition (EMT). In EMT, cells lose epithelial cell characteristics, become increasingly motile, and acquire the ability to invade surrounding tissue, enabling metastasis. A series of studies within the past year have nominated the HEF1 protein as an essential switch for pro-metastatic behavior in tumors. These studies have identified HEF1 as a component of a small "signature" of genes upregulated in metastasizing breast adenocarcinomas, shown that HEF1 is important for glioblastoma invasiveness, and determined that upregulation of HEF1 occurred in more than 30% of metastatic melanomas and was critical for the metastatic process. Since our group first described the HEF1 gene in 1996, we have worked continually to dissect the signaling role of this protein. This work has demonstrated a central role for HEF1 as a scaffolding protein or "hub" that enhances and coordinates the activity of multiple signaling cascades that promote cancer progression. In this proposal, we will address two basic questions: 1) How does absence or elevation of HEF1 overexpression condition classic EMT/metastatic responses, including expression of E-cadherin? and 2) Is elevated expression of HEF1 an important determinant of breast cancer metastasis? In specific experiments to answer these questions, Aim 1 will use mechanistic, cell-based assays to investigate how HEF1 regulates EMT. We will test the hypothesis that HEF1 promotes EMT by directly controlling the ability of E-cadherin to form stable structures at adherens junctions, and determine whether HEF1 is an essential intermediate in TGF-[unreadable] or ErbB2-induced EMT. Aim 2 will use genetically defined HEF1-/- mice to test the hypothesis that the ability to elevate HEF1 expression contributes significantly to breast cancer metastasis. We will cross HEF1-/- mice to two strains of transgenic mice (MMTV-PyVT and MMTV-neu) that develop primary breast tumors and lung metastases, and determine whether absence of HEF1 limits metastasis, and conditions TGF-[unreadable] and ErbB2-related signaling in tumor cells. Finally, Aim 3 will ask if elevated HEF1 predicts a metastatic profile in human breast tumors. The ultimate goal of these experiments is to improve the diagnosis and treatment of cancer in human patients.